Site-specific inhibitors of histone methyltransferase [HMTase] and process of preparation thereof

ABSTRACT

The present invention relates to compounds of structural formula I as site-specific inhibitors of histone methyltransferase, where R 1  and R 1  is —OCH 3  or —OH, R 2  and R 2  is —OH or —OAc; a process of isolating compound of structural formula Ia, where R 1  and R 1  is —OCH 3 , R 2  and R 2  is —OH; a process of preparation of compound of structural formula Ib, where R 1  and R 1  is —OCH 3 , R 2  and R 2  is —OAc; a process of preparation of compound of structural formula Ic, where R 1  and R 1  is —OH, R 2  and R 2  is —OH; and use of compounds of structural formula I for manufacture of a medicament for management of cancer and/or disease conditions in a subject in need thereof.

FIELD OF INVENTION

The present invention relates to the field of small molecule modulators.More particularly the invention relates to histone modifying enzymes,which may serve as lead compounds to develop anti-neoplastic andanti-HIV therapeutics. These molecules may also be useful to probe thefunction of HMTases in vivo.

The present invention relates to compounds as site-specific inhibitorsof histone methyltransferase and a process of preparation thereof.

BACKGROUND AND PRIOR ART

Eukaryotic genome is packaged into a highly dynamic chromatin structure,the unit of which is nucleosome, composed of four different corehistones octamer wrapped around by 146 by of DNA. The posttranslationalmodifications namely reversible acetylation, methylation,phosphorylation etc., of the tails of the core histones significantlycontribute to the dynamic structure-function organization of chromatinwhich in turn plays a crucial role in the different DNA-templatedphenomenon in the cell. Among these modifications recent focus onhistone methylation and its cellular function has gained a broadspectrum of interest. The various disorders where HMTases play asignificant role include cancers at the top of the list, followed bycardiovascular diseases, viral pathogenesis as well as multiplesclerosis.

OBJECTS OF THE PRESENT INVENTION

The principal object of the present invention is to obtain compounds ofstructural formula I as site-specific inhibitors of histonemethyltransferase.

Another main object of the present invention is to obtain compounds ofstructural formula I where R¹ and R₁ is —OCH₃ or —OH, R² and R₂ is —OHor —OAc.

Yet another object of the present invention is to obtain compounds ofstructural formula I as site-specific inhibitors of argininemethyltransferase, preferably CARM1.

Still another object of the present invention is to obtain a process ofisolating compound of structural formula Ia, where R¹ and R₁ is —OCH₃,R² and R₂ is —OH,

Still another object of the present invention is to obtain a process ofpreparation of compound of structural formula Ib, where R¹ and R₁ is—OCH₃, R² and R₂ is —OAc.

Still another object of the present invention is to obtain a process ofpreparation of compound of structural formula Ic, where R¹ and R₁ is—OH, R² and R₂ is —OH.

Still another object of the present invention is to synthesizePolyhydroxy derivatives of (2,3,7,8-tetrahydroxy[1]benzopyrano(5,4,3(de)[1] benzopyran5,10-dione).

Still another object of the present invention is to synthesize moreefficient and nontoxic derivatives of (2,3,7,8-tetrahydroxy[1]benzopyrano(5,4,3(de)[1]benzopyran5,10-dione).

Still another object of the present invention is to synthesize compoundswhich are inhibitors of Histone Methyltransferases.

Still another object of the present invention is to develop compoundswhich act as anti-cancerous and anti-HIV agents.

Still another object of the present invention is to understand the roleof RMTase in cellular function.

STATEMENT OF THE INVENTION

The present invention relates to compounds of structural formula I assite-specific inhibitors of histone methyltransferase, where R¹ and R₁is —OCH₃ or —OH, R² and R₂ is —OH or —OAc; a process of isolatingcompound of structural formula Ia, where R¹ and R₁ is —OCH₃, R² and R₂is —OH, the process comprising steps of: (a) obtaining husk ofpomegranate fruit by way of drying and powdering; (b) stirring thepowdered husk followed by filtering the precipitate to collect thefiltrate as semi-solid extract; (c) loading the semi-solid extract in acolumn and eluting the crude product with suitable solvent system; and(d) purifying crude product to obtain a fraction containing the compoundof structural formula Ia; a process of preparation of compound ofstructural formula Ib, where R¹ and R₁ is —OCH₃, R² and R₂ is —OAc, theprocess comprising steps of: (a) stirring the reaction mixturecontaining compound of structural formula Ia, acetic anhydride andsulphuric acid; (b) raising the temperature of reaction mixture followedby continued stirring to obtain reaction mass; (c) filtering thereaction mass and washing the precipitate to obtain residue; and (d)drying and hydrolyzing the residue to obtain a precipitate containingcompound of structural formula Ib; a process of preparation of compoundof structural formula Ic, where R¹ and R₁ is —OH, R² and R₂ is —OH, theprocess comprising steps of: (a) refluxing the reaction mixturecontaining compound of structural formula Ia and potassium hydroxidesolution in methanol; (b) cooling the refluxed reaction mixture toobtain reaction mass followed by its acidification to yield organicproduct; and (c) extracting the organic product with dichloromethane toobtain the compound of structural formula Ic; and use of compounds ofstructural formula I for manufacture of a medicament for management ofcancer and/or disease conditions in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds of structural formula I assite-specific inhibitors of histone methyltransferase, where R¹ and R₁is —OCH₃ or —OH, R² and R₂ is —OH or —OAc.

In another embodiment of the present invention, the compounds areisolated from pomegranate fruit.

In yet another embodiment of the present invention, the histonemethyltransferase is arginine methyltransferase, preferably CARM1.

The present invention also relates to a process of isolating compound ofstructural formula Ia, where R¹ and R₁ is —OCH₃, R² and R₂ is —OH, theprocess comprising steps of:

-   -   a) obtaining husk of pomegranate fruit by way of drying and        powdering;    -   b) stirring the powdered husk followed by filtering the        precipitate to collect the filtrate as semi-solid extract;    -   c) loading the semi-solid extract in a column and eluting the        crude product with suitable solvent system; and    -   d) purifying crude product to obtain a fraction containing the        compound of structural formula Ia.

In still another embodiment of the present invention, the solvent systemis methanol:water system.

The present invention also relates to a process of preparation ofcompound of structural formula Ib, where R¹ and R₁ is —OCH₃, R² and R₂is —OAc, the process comprising steps of:

-   -   a) stirring the reaction mixture containing compound of        structural formula Ia, acetic anhydride and sulphuric acid;    -   b) raising the temperature of reaction mixture followed by        continued stirring to obtain reaction mass;    -   c) filtering the reaction mass and washing the precipitate to        obtain residue; and    -   d) drying and hydrolyzing the residue to obtain a precipitate        containing compound of structural formula Ib.

In still another embodiment of the present invention, the temperature isslowly raised from about 100 degree celsius.

In still another embodiment of the present invention, the precipitate iswashed to remove unreacted acetic anhydride.

In still another embodiment of the present invention, the precipitate iswashed with organic solvent, preferably acetone.

In still another embodiment of the present invention, the residue ishydrolyzed with pyridine water.

The present invention also relates to a process of preparation ofcompound of structural formula Ic, where R¹ and R₁ is —OH, R² and R₂ is—OH, the process comprising steps of:

-   -   a) refluxing the reaction mixture containing compound of        structural formula Ia and potassium hydroxide solution in        methanol;    -   b) cooling the refluxed reaction mixture to obtain reaction mass        followed by its acidification to yield organic product; and    -   c) extracting the organic product with dichloromethane to obtain        the compound of structural formula Ic.

The present invention also relates to use of compounds of structuralformula I for manufacture of a medicament for management of cancer andfor disease conditions in a subject in need thereof.

In still another embodiment of the present invention, the compounds areinhibitors of histone methyltransferase.

In still another embodiment of the present invention, the histonemethyltransferase is arginine methyltransferase, preferably CARM1.

In still another embodiment of the present invention, the nature ofinhibition of compound is uncompetitive.

In still another embodiment of the present invention, the compounds areantiproliferative and antiangiogenic in nature.

In still another embodiment of the present invention, the compoundsmodulate the transcriptional activation of p53 responsive genes.

In still another embodiment of the present invention, the subject isanimal including human being.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

FIG. 1 a: Effect of LTK20 on Histone Modifying Enzymes (FilterbindingAssay)

Filterbinding Assay of LTK20 carried out with the Histoneacetyltransferases p300, PCAF and Histone methyltransferases CARM1, G9a.Lane1—histones alone, lane2—Enzyme control, lane3—DMSO control, lane4-5, LTK20 in increasing concentration. LTK20 inhibits CARM1 activity,but does not act on other enzymes.

FIG. 1 b: Effect of LTK20 on Histone Modifying Enzymes (Fluorography)

Gel Assay of LTK20 carried out with Histone acetyltransferases p300,PCAF and

Histone methyltransferases CARM1, G9a. Lane1—histones alone,lane2—Enzyme control, lane3—DMSO control, lane 4 & 5—LTK20 in increasingconcentration. LTK20 inhibits CARM1 activity, but does not act on otherenzymes.

FIG. 2: Binding Isotherm of Histone H3 with LTK20

Isothermal calorimetric Titration carried out with the protein histoneH3 and the ligand LTK20, which is the inhibitor. The binding isothermfits to the two sequential binding site model.

FIGS. 3 a and 3 b: Preferential Inhibition of CARM1 Mediated Methylation

Sitespecific inhibition of arginine methylation of histone H3 observedin the presence of LTK20 both in vitro and in vivo. H3R17 and H3R26 areboth CARM1 methylation sites on histone H3. The methylation of H3R17 isinhibited in the presence of the inhibitor LTK20 whereas the other siteis getting methylated.

FIGS. 3 c and 3 d: Immunofluorescence Analysis of LTK20 Treated HeLaCells

FIG. 4: Immunoblot Analysis with p21 Antibody

UV irradiated U2OS cells treated with or without inhibitors. The lysatewas probed with antibody against p21. p21 levels show an increase in thepresence of LTK20 as compared to the DMSO control.

The present invention relates to Polyhydroxy Derivatives of compound of(2,3,7,8-tetrahydroxy[1]benzopyrano(5,4,3(de)[1]benzopyran5,10-dione).

The present invention also relates to a process for the preparation ofpolyhydroxy derivatives of the compound (2,3,7,8-tetrahydroxy[1]benzopyrano(5,4,3(de)[1] benzopyran5,10-dione).

The present invention further relates to method of treating a diseasecondition selected from a group comprising cancer, cardiac hypertrophy,Acquired Immunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus(HIV) in a subject in need thereof, wherein said method comprises stepof administering pharmaceutically effective amount of polyhydroxyderivatives of compounds of(2,3,7,8-tetrahydroxy[1]benzopyrano(5,4,3(de)[1]benzopyran5,10-dione).

We report the inhibitor of HMTase, which has implications in tumorsuppressor function. The inhibitor is highly specific to the Argininemethyltransferase (RMTase) CARM1 with an IC50 of 20 μm, but has aminimal effect on the lysine methyltransferase G9a. Significantly, ithas no effect on the histone acetyltransferases, p300 and PCAF. Theinhibitor is also active in vivo at a concentration of 100 μm. Ourstudies also indicate the possibility of the inhibitor to be morespecific to one of the sites of methylation out of the three known CARM1methylation sites. The inhibitor and its derivatives will thereforeprove to be important lead compounds for many disorders. Incidentally,the RMTase inhibitor is a potent antiproliferative and antiangiogeniccompound. Furthermore, this compound has also been shown to be toxic tocancerous cells but has no effect on the normal cells. This compound andits derivatives would be useful as a biological probe to understand therole of RMTase in cellular function. Significantly we have found thatthe compound isolated from the natural source is more potent than thesynthetic compound.

The invention is further elaborated with the help of following examples.However, these examples should not be construed to limit the scope ofthe invention.

EXAMPLE 1 Purification of(2,3,7,8-tetrahydroxy[1]benzopyrano[5,4,3(de)[1]benzopyran5,10-dione)from pomegranate

Pomegranate fruits were washed and cleaned to yield their husks whichwere separated from seeds and juice. The husks were dried in thesunlight and powdered by mixer. Pomegranate husk powder (250 gram) wascharged into the four neck one liter flask fitted with magnetic stirrer,followed by addition of one liter of water and stirred for 24 hours. Theprecipitate was filtered with the help of a buchner funnel and thecollected filtrate was distilled up completely to give a semi solidbrown coloured component. This semisolid extract was divided into 10portions. Two portions were loaded into silica gel column of 180-200mesh with water:methanol as the solvent system. The column was elutedwith distilled water until the sugary pale yellow eluate was clear incolour. The crude product was eluted with 2:1 methanol:water system. Themethanol and water was distilled up completely with rotary evaporation.

Purification of Crude Product with Sephadex LH20

The sephadex LH20 column was pre-equilibrated with water:methanol (8:2)system. Crude product (3 gram) was loaded into the column. The columnwas eluted with increasing amount of methanol. Different fractions werecollected and spotted on TLC. Similar fractions were combined. Fivedifferent fractions were collected and named as fraction A, B, C, D, E.The fraction A was characterized as the molecule having inhibitoryactivity against the histone methyltransferase CARM1.

EXAMPLE 2 Derivatization of(2,3,7,8-tetrahydroxy[1]benzopyrano[5,4,3(DE)[1]benzopyran5,10-dione)

(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)was charged into the three necked flask fitted with magnetic stirrer,oil bath and thermo pocket, followed by addition of acetic anhydride (3ml) and a drop of sulphuric acid. The reaction mixture was stirred for10 minutes and the temperature was slowly raised from 100 degree Celsiusand stirred for one hour. The reaction mass was cooled to 20-25 degreecelsius and the reaction mass was filtered. The precipitate was washedwith 10 ml acetone to remove unreacted scetic anhydride. The residue wasdried and hydrolyzed with 2 ml of pyridine-water by heating briefly at115 degree celsius. After cooling the reaction mixture, the precipitateswere collected, washed with 500 ml of water and 3 ml of acetone anddried. This gave the derivative where R2—OAc is obtained.

(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE) (1gram) was charged into the 100 mc neck flask fitted with magneticstirrer and oil bath, followed by addition of potassium hydroxidesolution (5M) in methanol (20 ml). The reaction mixture was refluxed for8-10 hours. The reaction mixture was cooled to room temperature. Themethanol was distilled up completely and the reaction mass was acidifiedwith 2N HCl solution. The organic product was extracted with dichloromethane, followed by its complete removal and complete drying. This gaveR1-OH derivative of the parent compound.

General Formula: C₁₄H₁₈O₄R1R2, where R1—OCH₃ (LTK20), —OH (LTK54)

-   -   R2—OH (LTK20), —OAc (LTK51)

The designation LTK stands for L—Lingu and TK—Tapas Kundu. LTK20 is theparent compound(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN 5,10-DIONE)and therefore according to the general structure given R₁, R¹—OCH₃ andR₂ and R²—OH.

EXAMPLE 3 In vitro Characterization of (2,3,7,8-tetrahydroxy[1]benzopyrano[5,4,3(DE)[1]benzopyran5,10-dione) and its derivatives

Filterbinding and gel assay of(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)against CARM1/G9a/p300/PCAF

The inhibitory activity of the molecule(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)was assayed against the histone acetyltransferases p300 and PCAF, lysinemethyltransferase G9a, arginine methyltransferase CARM1 using HeLacorehistones as the substrate according to the standard HAT assay andHMTase assay.(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)was identified as a specific inhibitor of the arginine methyltransferaseCARM1 with an IC50 of 20 μM, both by filterbinding (FIG. 1 a) andfluorography assays (FIG. 1 b).

Filterbinding assay of LTK51 and LTK54 (derivatives of(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE))against CARM1/G9a

The inhibitory activity of the derivatives LTK51 and LTK54 were assayedagainst, lysine methyltransferase G9a, arginine methyltransferase CARM1using HeLa core histones as the substrate according to the standardHMTase assay. The derivatives containing R1—OH and R2—OAc groups werefound to be not active, indicating that R1-OCH₃ and R2—OH groups areessential for the inhibitory activity.

EXAMPLE 4 Kinetic Analysis of the Pattern of Inhibition

Since the inhibitor is specific to CARM1 mediated methylation ofhistones which is directed to the histone H3, histone H3 was used as thesubstrate for the kinetic analysis. Initially, the inhibition wasconfirmed using the recombinant histone as the substrate. CARM1methylation reaction was carried out using increasing concentration ofhistone H3 and the methyl group donor, tritiated SAM keeping the enzymeconcentration constant. Two different inhibitor concentrations wereassayed. The kinetic analysis revealed the pattern of(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)mediated CARM1 methylation inhibition is Uncompetitive.

Substrate Binding Data by ITC Experiment

The pattern of inhibition is uncompetitive indicating the mode ofinhibition is via the Enzyme-Substrate complex, the binding of theinhibitor has been characterized by Isothermal calorimetric Titrationwherein either the substrate (histone H3) or enzyme (CARM1) was taken inthe cell and titrated against the inhibitor(2,3,7,8TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE).The data shows clearly that there is no binding of the molecule to theenzyme, rather it binds to the substrate at two sequential binding sites(FIG. 2).

Site Specific Inhibition Shown by Western Analysis

In vitro CARM1 methylation reaction was carried out in the presence orabsence of inhibitors, and probed with antibodies against the specificCARM1 methylation sites on histone H3, R17 and R26. Site specificinhibition of H3R17 was observed indicating that(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE) isa novel site specific inhibitor (FIG. 3 a).

EXAMPLE 5 Cellular Effects of (2,3,7,8-tetrahydroxy[1]benzopyrano[5,4,3(DE)[1]benzopyran5,10-dione) In vivo Characterization

Site Specific Inhibition Shown by Western Analysis

In vivo characterization was done by treating HeLa cells with themolecule at 50 μM concentration for a period of 24 hours, and the acidextracted histones were probed with antibodies against CARM1 methylationsites on histone H3, R17 and R26. Site specific inhibition of H3R17 wasobserved indicating that(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE) isa novel site specific inhibitor, even in the in vivo context (FIG. 3 b).

Site Specific Inhibition Shown by Immunofluorescence Analysis

In vivo characterization was done by treating HeLa cells with themolecule at 50 μM concentration for a period of 24 hours, and the cellswere probed with antibodies against CARM1 methylation sites on histoneH3, R17 and R26 and were visualized by anti rabbit alexa fluor 488antibody. Site specific inhibition of H3R17 was observed indicating that(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE) isa novel site specific inhibitor, even in the in vivo context (FIG. 3 cand FIG. 3 d).

EXAMPLE 6 Modulation of p53 and p21 Protein Levels in U2OS Cells byWestern Analysis

Physiological effect of the inhibitor was characterized by selecting thep53 system, since CARM1 mediated histone methylation is known tocooperatively enhance the transcriptional activation of p53 responsivegenes. Luciferase reporter assays did not work because of theantioxidant property of(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE).U2OS, which is a p53 positive cell line was used for the assay, and ontreatment with molecule at 50 μM concentration for a period of 24 hours,p53 levels were altered. Since, the p53 responsive genes had to beassayed, p21 was selected

(There is no apoptosis, therefore the apoptosis genes were notselected). UV irradiated cells were treated with(2,3,7,8-TETRAHYDROXY[1]BENZOPYRANO[5,4,3(DE)[1]BENZOPYRAN5,10-DIONE)for a period of 24 hours and the lysate was probed with antibody againstp21. There is an increase in p21 levels on treatment (FIG. 4). Thisclearly indicates a possible implication of this molecule in the tumorsuppressor p53 mediated transcriptional activation.

1. Isolated compounds of structural formula I as site-specificinhibitors of histone methyltransferase, where R¹ and R₁ is —OCH₃ or—OH, R² and R₂ is —OH or —OAc


2. The compounds as claimed in claim 1, wherein the compounds areisolated from pomegranate fruit.
 3. The compounds as claimed in claim 1,wherein the histone methyltransferase is arginine methyltransferase,preferably CARM1.
 4. A process of preparing an isolated compound ofstructural formula Ia, where R¹ and R₁ is —OCH₃, R² and R₂ is —OH, theprocess comprising steps of:

a. obtaining husk of pomegranate fruit by way of drying and powdering;b. stirring the powdered husk followed by filtering the precipitate tocollect the filtrate as semi-solid extract; c. loading the semi-solidextract in a column and eluting the crude product with suitable solventsystem; and d. purifying crude product to obtain a fraction containingthe compound of structural formula Ia.
 5. The process as claimed inclaim 4, wherein the solvent system is methanol: water system.
 6. Aprocess of preparing an isolated compound of structural formula Ib,where R¹ and R₁ is —OCH₃, R² and R₂ is —OAc, the process comprisingsteps of

a. stirring the reaction mixture containing compound of structuralformula Ia, acetic anhydride and sulphuric acid; b. raising thetemperature of reaction mixture followed by continued stirring to obtainreaction mass; c. filtering the reaction mass and washing theprecipitate to obtain residue; and d. drying and hydrolyzing the residueto obtain a precipitate containing compound of structural formula Ib. 7.The process as claimed in claim 6, wherein the temperature is slowlyraised from about 100 degree celsius.
 8. The process as claimed in claim6, wherein the precipitate is washed to remove unreacted aceticanhydride.
 9. The process as claimed in claim 6, wherein the precipitateis washed with organic solvent, preferably acetone.
 10. The process asclaimed in claim 6, wherein the residue is hydrolyzed with pyridinewater.
 11. A process of preparing an isolated compound of structuralformula Ic, where R¹ and R₁ is —OH, R² and R₂ is —OH, the processcomprising steps of:

a. refluxing the reaction mixture containing compound of structuralformula Ia and potassium hydroxide solution in methanol; b. cooling therefluxed reaction mixture to obtain reaction mass followed by itsacidification to yield organic product; and c. extracting the organicproduct with dichloromethane to obtain the compound of structuralformula Ic.